Lutein is a light yellow pigment belonging to the xanthophyll group. It is characterized by hepatoprotective, neuroprotective, anti-inflammatory and antioxidant activity. This paper present the optimisation of lutein extraction by examining the influence of different extraction mixtures, the influence of ultrasound, ratio of dry matter (sample) and extraction mixture, sample particle size and extraction time on the success of lutein extraction from the complex sample. The highest lutein content was determined in grinded samples, the direct ultrasonic extraction proved to be better, and the extraction mixture of methanol:acetone= 1:1 showed best results. The best dry matter to sample ratio was 1:20 in Method 1 in which methanol: acetone= 1:1 was used as the extraction mixture, while in Method 4 it was 1:40 in which hexane was added as the final solvent with 1 hour of sample resting in the dark place. Method 4, which proved to be the most successful, was used to analyze the real samples. This method was used to determine the content of lutein in eight types of food for laying hens. The highest lutein content was determined in food samples enriched with lutein, selenium, omega-3 fatty acids and vitamin E (75.314 mg / kg of sample), while the lowest lutein content was determined in food available on the market that was not mixed with cereals, 8.482 mg / kg of sample.